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Chronic loss of noradrenergic tone produces arrestin2mediated cocaine hypersensitivity and alters cellular d2 responses in the nucleus accumbens

bs_bs_banner Addiction Biology Chronic loss of noradrenergic tone produces
β-arrestin2-mediated cocaine hypersensitivity and
alters cellular D2 responses in the nucleus accumbens
Meriem Gaval-Cruz1*, Richard B. Goertz2*, Daniel J. Puttick1, Dawn E. Bowles3,
Rebecca C. Meyer4, Randy A. Hall4, Daijin Ko5, Carlos A. Paladini2 & David Weinshenker1
Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA1, Department of Biology, Neurosciences Institute, University of Texasat San Antonio, San Antonio, TX, USA2, Department of Surgery, Duke University School of Medicine, Durham, NC, USA3, Department of Pharmacology,Emory University School of Medicine, Atlanta, GA, USA4 and Department of Management Science and Statistics, University of Texas at San Antonio, San Antonio,TX, USA5 Cocaine blocks plasma membrane monoamine transporters and increases extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT). The addictive properties of cocaine are mediated primarily by DA, while NE and 5-HT play modulatory roles. Chronic inhibition of dopamine β-hydroxylase (DBH), which converts DA to NE,increases the aversive effects of cocaine and reduces cocaine use in humans, and produces behavioral hypersensitivity to cocaine and D2 agonism in rodents, but the underlying mechanism is unknown. We found a decrease in β-arrestin2(βArr2) in the nucleus accumbens (NAc) following chronic genetic or pharmacological DBH inhibition, andoverexpression of βArr2 in the NAc normalized cocaine-induced locomotion in DBH knockout (Dbh −/−) mice. TheD2/3 agonist quinpirole decreased excitability in NAc medium spiny neurons (MSNs) from control, but not Dbh −/−animals, where instead there was a trend for an excitatory effect. The Gαi inhibitor NF023 abolished the quinpirole-induced decrease in excitability in control MSNs, but had no effect in Dbh −/− MSNs, whereas the Gαs inhibitor NF449restored the ability of quinpirole to decrease excitability in Dbh −/− MSNs, but had no effect in control MSNs. Theseresults suggest that chronic loss of noradrenergic tone alters behavioral responses to cocaine via decreases inβArr2 and cellular responses to D2/D3 activation, potentially via changes in D2-like receptor G-protein couplingin NAc MSNs.
Cocaine, D2 receptor, dopamine, dopamine β-hydroxylase, mice, norepinephrine.
Correspondence to: David Weinshenker, Department of Human Genetics, Emory University School of Medicine, Whitehead 301, 615 Michael St., Atlanta, GA 30322, USA. E-mail: [email protected] Shandilya & Kundu 2011), alters the subjective effects of cocaine and reduces cocaine use in humans (Stanley Dopamine β-hydroxylase (DBH) is the enzyme that et al. 1997; Gaval-Cruz & Weinshenker 2009) (K.
converts dopamine (DA) to norepinephrine (NE) in Cunningham, pers. comm.). Genetic (DBH knockout; Dbh noradrenergic neurons, thereby controlling NE produc- −/−) or pharmacological (disulfiram, nepicastat) DBH tion and the DA/NE ratio (Weinshilboum 1978). DBH is inhibition produces hypersensitivity to cocaine-induced of clinical interest in cocaine dependence because: (1) locomotion, stereotypy, place preference and place aver- polymorphisms in the human DBH gene that are associ- sion in mice; it also enhances the discriminative stimulus ated with reduced serum DBH enzymatic activity lead to effects of cocaine and attenuates cocaine-, cue- and greater cocaine-induced paranoia (Cubells et al. 2000; stress-induced reinstatement of cocaine seeking in Kalayasiri et al. 2007); and (2) inhibition of DBH by the rats (Schank et al. 2006; Schroeder et al. 2010, 2013; alcoholism medication, disulfiram, or the selective DBH Gaval-Cruz et al. 2012; Manvich, Depoy & Weinshenker inhibitor, nepicastat (Stanley et al. 1997; Kapoor, *MGC and RBG contributed equally to this work.
2014 Society for the Study of Addiction Addiction Biology Meriem Gaval-Cruz et al. Because Dbh −/− mice are hypersensitive to the D2/3 mice for the experiments (Thomas, Matsumoto & Palmiter agonist, quinpirole, but not the D1 agonist, SKF81297, 1995; Thomas et al. 1998). Comparable numbers of male cocaine hypersensitivity would appear to be mediated by and female knockouts were used for each experiment, and alterations in the D2 pathway (Weinshenker et al. 2002; sex-matched Dbh +/− littermates were used as controls.
Schank et al. 2006). These phenotypes are likely driven Although the studies were not powered sufficiently to by compensatory responses in DA signaling following the rigorously detect sex differences, no obvious ones chronic decrease in extracellular DA availability when were observed. The Dbh +/− mice were used as controls noradrenergic excitatory drive on the mesocorticolimbic because their brain catecholamine levels and behaviors system is missing. We initially reported an increase in the are indistinguishable from wild-type (Dbh +/+) mice abundance of high-affinity state D2 receptors in the (Thomas et al. 1998; Bourdelat-Parks et al. 2005; striatum of Dbh −/− mice, which could explain the Mitchell et al. 2006). Some wild-type C57BL/6J mice cocaine and D2 hypersensitivity (Schank et al. 2006).
(Jackson Laboratory, Bar Harbor, ME, USA) were also used However, subsequent work failed to confirm this finding as controls for the electrophysiology experiments.
(Skinbjerg et al. 2010), suggesting a contribution from All animals were treated in accordance with the downstream signaling molecules. Indeed, the behavioral National Institutes of Health Intramural Animal Care and alterations in Dbh −/− mice were accompanied by a rise in Use Program guidelines. The experiments described in this striatal pERK and ΔFosB protein levels (Rommelfanger article followed the UTSA and Emory University Division et al. 2007).
of Animal Resources' Guide for the Care and Use of Labo- The goals of the present study were to determine the ratory Animals and were approved by the UTSA and molecular and cellular mechanisms behind the D2- and Emory Institutional Animal Care and Use Committee.
psychostimulant-induced hypersensitivity that follow chronic DBH inhibition. First, we found a decrease of Chronic nepicastat treatment
β-arrestin2 (βArr2), a protein involved in D2 desensitiza- Nepicastat was administered to Dbh +/− mice via daily i.p.
tion and signaling (Beaulieu & Gainetdinov 2011), in the injections (Western blots) or osmotic minipumps (locomo- nucleus accumbens (NAc) of Dbh −/− mice and mice tor activity). For the i.p. administration, Dbh +/− mice treated chronically with nepicastat. We next used viral- received vehicle or nepicastat (50 mg/kg, i.p. × 3, each mediated overexpression to determine whether increas- injection spaced 2 hours apart) for 5 consecutive days.
ing βArr2 levels in the NAc could normalize cocaine- This dosing regimen reduces brain NE levels by ∼75 per- induced behavior in Dbh −/− mice. Finally, we assessed cent and produces cocaine hypersensitivity (Gaval-Cruz electrophysiological responses to quinpirole in medium et al. 2012). Mice were euthanized by CO2 asphyxiation 11 spiny neurons (MSNs) from the NAc of control and Dbh days later, and their brains were removed, dissected on ice −/− mice in the presence and absence of Gαi and Gαs and stored at −80°C. For the minipump administration, nepicastat was dissolved in 50 percent saline and 50 percent dimethyl sulfoxide and loaded into Alzet osmotic minipumps (Model #2004, 0.25 μl/hour, 28 days; MATERIALS AND METHODS
Durect, Cupertino, CA, USA) to achieve a dose of 50 mg/ kg/day. All pumps were placed in a sterile 37°C saline bath for 1 day before implantation. Mice were anesthe- Adult control (Dbh +/−) and Dbh −/− mice were generated tized with isoflurane, and minipumps were implanted in as previously described (Thomas et al. 1998; Schank the intraperitoneal cavity. Buprenorphine (2.5 mg/kg, et al. 2006). Dbh −/− males were bred to Dbh +/− females.
s.c.) was given immediately after surgery. Cocaine- Pregnant Dbh +/− mice were given the AR agonists induced locomotion was recorded 21 days after pump isoproterenol and phenylephrine (20 μg/ml each) + vitamin C (2 mg/ml) from E9.5-E14.5, and L-3,4- dihydroxyphenylserine (2 mg/ml) + vitamin C (2 mg/ml) from E14.5 birth in their drinking water to rescue the embryonic lethality associated with the homozygous Dbh Mice were placed in locomotion recording chambers −/− mutation. Because of this treatment, NE and epineph- (transparent Plexiglas cages placed into a rack with seven rine were present in Dbh −/− animals before but not after infrared photobeams spaced 5 cm apart; San Diego birth. They were maintained on a mixed C57BL/6J and Instruments Inc., La Jolla, CA, USA) and allowed to 129SvEv background and group housed, and food and habituate for 30 minutes before receiving a single injec- water were available ad libitum throughout the course of tion of cocaine (10 or 15 mg/kg, i.p.). Novelty-induced the study. Both sexes were used due to the extreme meas- locomotion was defined as ambulations during the first ures required to breed sufficient numbers of knockout 10 minutes of the habituation period. Ambulations 2014 Society for the Study of Addiction Addiction Biology Cocaine response in DBH mice (consecutive beam breaks) were recorded for an addi- 1:1000; Cell Signaling, CS9271); Akt (anti-mouse; tional 1–2 hours following drug administration.
1:500; Santa Cruz Biotechnology, SC5298); pGSK3β-Ser9 (anti-rabbit; 1:1000; Cell Signaling, CS9322); pGSK3β (anti-rabbit; 1:1000; Cell Signaling, CS9315);FosB (anti-rabbit; 1:1000; Cell Signaling, CS9890).
Mouse brain tissue was homogenized in 500 μlharvest βArr2 viral vectors
piperazineethanesulfonic acid (HEPES), 50 mM NaCl, 5 mM ethylenediaminetetraacetic acid, pH 7.4, supple- The original βArr2 plasmid (rat sequence) was obtained mented with protease inhibitors] using a sonicator.
from Sudha Shenoy in the laboratory of Dr. Robert Laemmli sample buffer containing sodium dodecyl sulfate, Lefkowitz. The Duke Neurotransgenic Laboratory then β-mercaptoethanol, glycerol, Tris-Cl and bromophenol removed the βArr2 open reading frame, and the insert blue was added to samples after measuring protein con- was cloned into a pCMVShuttle plasmid (AdEasy System, centrations with a bicinchoninic acid assay (Thermo Stratagene, Santa Clara, CA, USA). The AdEasy βArr2 Fisher Scientific, Rockford, IL, USA). Samples were recombinant plasmid was generated per Stratagene resolved by sodium dodecyl sulfate–polyacrylamide gel instructions, and the βArr2 adenoviral vector was electrophoresis on 4–20 percent Tris-glycine precast gels expanded and purified. The viruses were harvested with a followed by transfer to nitrocellulose membranes. Follow- titer of 2 × 1012/μl (βArr2) and 5 × 109/μl [green fluo- ing transfer, membranes were incubated with Ponceau rescent protein (GFP) control].
staining to assess even protein loading, then rinsed with distilled water. Membranes were then incubated in block- βArr2 viral infusions
ing buffer (10 mM HEPES, 50 mM NaCl, 1 percent Tween- (n = 16 for each treatment group: βArr2 20, 2 percent dry milk, pH 7.4, for most antibodies; 1X overexpression adenovirus and GFP adenovirus) were TBS, 0.1 percent Tween-20 with 5 percent w/v non-fat anesthetized using isoflurane and placed in a stereotaxic dry milk, for pAKT, GSK3β and pGSK3β) for 30 minutes, frame with a nose bar. The animal's scalp was opened and and then incubated with primary antibody overnight at bregma and lambda aligned to flat-skull position. The 4°C. The primary incubation buffer was the same as block- stereotaxic arm was then lowered to the NAc core.
ing buffer for all antibodies except pAKT, GSK3β and The core subregion was chosen because it has been impli- pGSK3β. For these, the primary incubation buffer was 1X cated in cocaine-induced locomotion and behavioral TBS, 0.1 percent Tween-20 with 5 percent bovine serum sensitization to cocaine. The anteroposterior (AP) and albumin (BSA). The membranes were washed three times mediolateral (ML) coordinates of the NAc core in relation in blocking buffer and incubated with either a fluorescent to bregma were AP = 1.4 mm and ML = ±1.0 mm, and (1:10 000) or horseradish peroxidase-conjugated second- a small hole was drilled in the skull at these coordinates.
ary (1:4000) antibody (Invitrogen, Carlsbad, CA, USA) for A 5-μl Hamilton microsyringe was lowered to target 30 minutes, washed three more times, and then visualized the NAc core (dorsoventral coordinate = −4.2 mm). The using either the Odyssey imaging system (Li-Cor, Lincoln, 26-gauge beveled tip of the Hamilton needle was NE, USA) or via enhanced chemiluminescence reagent precoated with 2 percent BSA prior to loading the virus to (Thermo Fisher Scientific), followed by exposure to film.
prevent molecular interactions between the syringe and Membranes were stripped for 20 minutes at 37°C and 10 the viral vectors. Animals received 1 μl of virus per side, minutes at room temperature with stripping buffer and injected at a rate of 0.2 μl/minute, and the needle re-probed for α-actin to confirm equal loading of samples.
remained in place for 5 minutes after the injection and Blots were analyzed by densitometry using ImageJ Soft- removed slowly. The skin was glued together using ware (National Institutes of Health, Bethesda, MD, USA).
Vetbond tissue glue (Henry Schein, Roswell, GA, USA).
A mean density value was calculated for the ‘control' All animals received meloxicam (0.5 mg/kg) for postop- group (i.e. Dbh +/− mice were the control for Dbh −/− mice, erative pain and water/liquid ibuprofen (0.1 mg/ml).
vehicle was the control for nepicastat), and data were Ten days after the infusion of βArr2 overexpres- expressed as % control.
sion and GFP control vectors, all mice were placed in locomotor chambers, and their basal locomotion was recorded for 30 minutes before receiving an injection of The antibodies used and their working dilutions were as cocaine (15 mg/kg, i.p.), and cocaine-induced locomo- follows: βArr2 (anti-rabbit; 1:2500; Cell Signaling tion was recorded for 2 hours. Mice were anesthetized Technology, Danvers, MA, USA, CS3857); α-actin and transcardially perfused with saline and 4 percent (anti-mouse; 1:1000; Santa Cruz Biotechnology, Santa paraformaldehyde 24–48 hours later, their brains were Cruz, CA, USA, SC58671); pAkt-Ser473 (anti-rabbit; removed, stored in 4 percent paraformaldehyde for 4 2014 Society for the Study of Addiction Addiction Biology Meriem Gaval-Cruz et al. days, and then transferred to 30 percent sucrose. Brains (< 350 MΩ), and delayed spiking upon current injection.
were sectioned and stained with antibodies against GFP Drugs were applied to the slice by superfusion at the indi- or βArr2, and expression in the NAc was assessed. Three cated concentration. All experiments were performed in mice that received the βArr2 virus and two mice that the presence of 5 μM NBQX (AMPA antagonist), 25 μM received GFP virus were removed from the analysis due to D-APV (NMDA antagonist), 100 μM picrotoxin (GABAa incorrect placement of viral infusion.
antagonist) and 10 μM SCH 23390 (D1 antagonist). Thedrug NF023 (10 μM) was applied internally. For NF 449 Electrophysiological recordings of NAc neurons
(1 μM) application, the slices were incubated in the Gαs C57BL/6J, Dbh +/− and Dbh −/− mice were used for antagonist for 1 hour prior to recording, and then con- electrophysiological recordings. C57BL/6J mice were tinuously exposed to NF449 throughout the recording used to: (1) confirm that Dbh +/− mice NAc MSNs were process. All drugs were obtained from Tocris Bioscience similar to wild-type NAc MSNs; and (2) increase the (Bristol, UK) or Sigma-Aldrich (St. Louis, MO, USA). In number of cells in a few experiments when not enough current-clamp configuration, current was injected for appropriately sex- and age-matched Dbh +/− control 200 ms at 100-pA step intervals (100–500 pA) with 5 animals were available. Mice were anesthetized with a seconds between each pulse, until the cell was depolarized lethal dose of isoflurane and decapitated. The brains were and spikes were evoked. An input/output curve was quickly removed and placed into an ice-cold, oxygenated obtained under baseline conditions before and after cutting solution containing (in mM): 110 choline Cl, 2.5 superfusing 10 ml of a 5 μM solution of quinpirole for KCl, 1.25 NaH2PO4, 4 MgCl2, 2 CaCl2, 10 dextrose, 25 approximately 5 minutes. Action potentials were detected NaHCO3, 1.3 ascorbic acid, 2.4 sodium pyruvate and using an amplitude threshold, and spike frequency was 0.05 glutathione. Parasagittal brain slices containing the calculated as the reciprocal of the interspike interval.
NAc (250 μm) were cut using a vibrating tissue slicer(Microm HM 650V, Thermo Fisher Scientific). The slices were then transferred to an incubation chamber contain- Western blot data were analyzed by t-test using GraphPad ing warm (35°C) artificial cerebral spinal fluid (ACSF) for Prism 6.0 (La Jolla, CA, USA) for Macintosh (Apple, 1 hour prior to recordings, and then stored at room tem- Cupertino, CA, USA). Behavioral data were analyzed by perature. The slices were transferred to a recording two-way repeated measures ANOVA (RMANOVA), fol- chamber for the experiments, where they were sub- lowed by Bonferroni post hoc tests, where appropriate, merged in oxygenated ACSF. The ACSF was equilibrated using Prism. Electrophysiological data were analyzed by with 95 percent O2-5 percent CO2, had a pH of 7.2, and RMANOVA with a generalized estimating equation (GEE).
contained (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 In our data, the number of observations differed between MgCl2, 2 CaCl2, 10 dextrose, 25 NaHCO3, 1.3 ascorbic different current steps across cells. Because of the unbal- acid and 2.4 sodium pyruvate. The slices were superfused anced design, the classic RMANOVA was therefore not an with 34–36°C ACSF at a rate of 2 ml/minute.
appropriate test. We used the RMANOVA with a GEE The cells were visualized using gradient contrast illu- approach with an exchangeable correlation structure to mination through a 40X water-immersion lens attached take into account the unbalanced design, as well as cor- to an Olympus BX51 (Olympus, Center Valley, PA, USA) related observations. For each test, GEE uses a robust test upright microscope. Patch pipettes were pulled from (Wald χ2 test based on robust variance estimators) for borosilicate glass (o.d. 1.5 mm, i.d. 0.84 mm) using a each effect. These analyses were performed using R P-97 Flaming/Brown electrode puller (Sutter Instru- ments, Novato, CA USA). Pipettes were filled with a solu- tion containing (in mM): 138 K-gluconate, 10 HEPES, 0.0001 CaCl2, 0.2 ethylene glycol tetraacetic acid, 4 NaATP, 0.4 NaGTP and 2 MgCl2, with an osmolarity of Dbh −/− mice have decreased βArr2 in the NAc
270–275 mOsm and adjusted to a pH of 7.3 with potas- sium hydroxylase (KOH). Recordings were made using a We showed previously that ΔFosB, which is induced in MultiClamp 700B amplifier (Molecular Devices, Sunny- the NAc by chronic drug exposure and is known to vale, CA, USA). Signals were digitized at 15–30 kHz and promote psychostimulant-induced behaviors (Kelz et al.
saved to a hard drive for analysis using the software 1999), is elevated in the striatum of drug-naïve Dbh −/− program AxoGraph X (AxoGraph Scientific, Berkeley, CA, mice (Rommelfanger et al. 2007). As part of a larger survey to identify potential upstream mediators of the Spiny neurons in the NAc core were identified as cocaine hypersensitivity that follows chronic DBH inhibi- having the following properties: a hyperpolarized mem- tion, we found that Dbh −/− mice had significantly less brane potential (< −70 mV), a low input resistance βArr2 in the NAc (t6 = 3.493, P < 0.05) (Fig. 1a). Besides 2014 Society for the Study of Addiction Addiction Biology

Cocaine response in DBH mice β-arrestin2/actin ratio Dbh +/–
Dbh –/–
0 0 30 60 90 120 150 180 210 240 270 300 330 360
βarrestin-2/actin ratio pAkt/tAkt ratio (% control) Dbh +/–
Dbh –/–
ΔfosB/actin ratio Dbh +/–
Dbh –/–
Figure 1 Behavioral and neurochemical phenotypes of mice with chronic DBH deficiency. Western blot data (mean ± SEM above,
representative blot below) for (a) βArr2 : actin ratio, (b) protein kinase B (Akt; phospho : total ratio), and (c) glycogen synthase kinase-3β
(GSK-3β; phosphor : total ratio) in the nucleus accumbens (NAc) of Dbh +/− and Dbh −/− mice (n = 8 per group). *P < 0.05 compared
with Dbh +/− mice. (d) Cocaine-induced (10 mg/kg, i.p.) locomotion, (e) βArr2 : actin ratio, and (f) ΔFosB : actin ratio in the NAc of Dbh
+/− control mice treated chronically with the selective DBH inhibitor nepicastat. *P < 0.05, **P < 0.01, ****P < 0.0001 compared with
vehicle
its role in G-protein-coupled receptor desensitization, Nepicastat-treated mice are hypersensitive to cocaine
βArr2 can signal through a protein kinase B/glycogen and have decreased βArr2 and increased ΔFosB
synthase kinase-3β (Akt/GSK3β) pathway (Del'guidice, in the NAc
Lemasson & Beaulieu 2011); however, we detected no genotype differences in the proportion of phosphorylated We next determined whether the cocaine hypersensitivity Akt and GSK3β proteins compared with the total protein observed in Dbh −/− mice could be mimicked by chronic levels when comparing Dbh −/− mice to control Dbh +/− pharmacologic DBH inhibition in control mice. Dbh +/− mice (Fig. 1b & c).
mice with normal NE content that received chronic 2014 Society for the Study of Addiction Addiction Biology Meriem Gaval-Cruz et al. nepicastat (via osmotic minipump or daily i.p. injections) Dbh −/− mice overexpressing GFP or βArr2 had decreased had no change in locomotion induced by a novel environ- novelty-induced locomotion compared with Dbh +/− con- ment, but displayed increased cocaine-induced stereo- trols with normal NE content at the 10-minute time et al.
point. As expected, Dbh −/− mice that were infused with (Fig. 1d), reminiscent of Dbh −/− mice (Weinshenker the GFP virus were hypersensitive to cocaine-induced et al. 2002; Schank et al. 2006; Gaval-Cruz et al. 2012).
locomotion compared with mice with normal NE Two-way ANOVA revealed a main effect of content. By contrast, overexpression of βArr2 in the NAc (F11,110 = 17.55, P < 0.0001) and a treatment × time core of Dbh −/− mice completely normalized their cocaine interaction (F11,110 = 2.64, P < 0.01). Post hoc tests response (Fig. 2d). Two-way ANOVA revealed a main showed that peak cocaine-induced locomotion was sig- effect of time (F11,286 = 9.62, P < 0.0001), genotype nificantly enhanced by chronic nepicastat administra- (F2,26 = 5.20, P < 0.05), and a time × genotype interac- tion. Acute DBH inhibition, in contrast, does not tion (F22,286 = 2.88, P < 0.0001). Post hoc tests showed augment cocaine responses and can even inhibit them that Dbh −/− mice overexpressing GFP displayed (Maj, Przegalinski & Wielosz 1968; Haile et al. 2003; increased locomotion compared with Dbh +/− mice and Schroeder et al. 2013). These results indicate that the Dbh −/− overexpressing βArr2 at the 20-, 30- and hypersensitivity to psychostimulants seen in Dbh −/− 40-minute time points following cocaine administration, mice cannot be attributed to developmental alterations whereas there were no apparent differences between Dbh produced solely by DBH knockout, but likely results from +/− mice and Dbh −/− mice overexpressing βArr2 in the downstream changes in the signaling pathways that NAc at any time point. These results suggest that the occur following prolonged deficits in NE.
cocaine hypersensitivity conferred by chronic DBH inhi- Because Dbh −/− mice have decreased βArr2 in the bition is mediated, at least in part, by reduced βArr2 NAc and increased ΔFosB in the striatum, we measured levels in the NAc.
the relative levels of these proteins in the NAc of control mice following chronic treatment with nepicastat. We Dbh −/− NAc MSNs have aberrant responses
found that nepicastat-treated mice had decreased βArr2 (t14 = 3.49, P < 0.01; Fig. 1e) and increased ΔFosB(t14 = 2.69, P < 0.05; Fig. 1f), confirming that genetic To uncover the cellular underpinnings of the D2 and and pharmacological inhibition of NE synthesis produces cocaine hypersensitivity following chronic DBH inhibi- similar alterations in DA signaling proteins in the ventral tion, we measured the spike frequency of trains of action potentials elicited by the injection of current steps in NAc MSNs both at baseline and following bath application of quinpirole (5 μM) from control and Dbh −/− mice. The Overexpression of βArr2 in the NAc reverses cocaine
control group consisted of both Dbh +/− and wild-type hypersensitivity in Dbh −/− mice
C57Bl/6J mice because results comparing baseline and While ΔFosB is induced in the NAc by chronic drug expo- quinpirole responses in these groups were not signifi- sure and is known to promote psychostimulant-induced cantly different [n = 22 Dbh −/−, 24 C57Bl/6J; χ2 behaviors (Kelz et al. 1999), the role of βArr2 is less clear.
(d.f. = 1) = 0.1, P = 0.75] (Fig. 3). We found no genotype To determine whether the decreased βArr2 in the NAc of differences between control and Dbh −/− mice in baseline mice with chronic NE deficiency contributes to their firing rate in untreated MSNs (F4,160 = 0.43; P = 0.79; behavioral hypersensitivity to cocaine, we overexpressed data not shown), and activation of D2 receptors by GFP or βArr2 in the NAc of Dbh −/− mice using quinpirole did not significantly change the input resist- adenoviral vectors and assessed novelty- and cocaine- ance or the resting membrane potential in either control induced locomotor activity. High levels of or Dbh −/− mice (control ΔRin = 5.84 ± 4.28 MΩ, Dbh −/− immunoreactivity were evident along the needle track ΔRin = 6.57 ± 8.55 MΩ, P = 0.54; control ΔVrest = −0.71 and in both the core and shell subregions (Fig. 2a), and ± 1.80 mV, Dbh −/− ΔVrest = −1.36 ± 1.04 mV, P = 0.43).
βArr2 protein levels were doubled in the Dbh −/− NAc As reported previously and expected for a Gαi/o-coupled as assessed by Western blot (Fig. 2b) 7–10 days fol- receptor (Surmeier & Kitai 1993; Zamponi & Snutch lowing viral vector injection, indicating that βArr2 1998; Yasumoto et al. 2002; Perez, White & Hu 2006), overexpression was achieved and that the antibody we activation of D2 receptors by quinpirole (5 μM) reduced used was detecting βArr2. βArr2 overexpression had no evoked mean spike frequency in MSNs from control effect on the reduced novelty-induced locomotor activity animals [χ2 (d.f. = 1) = 5.2, P = 0.02] (Fig. 4a & c).
of Dbh −/− mice (Fig. 2c). Two-way ANOVA revealed a However, the D2-mediated reduction in MSN excitability main effect of time (F2,66 = 99.44, P < 0.0001) and geno- seen in control mice was absent in MSNs recorded from type (F2,33 = 4.46, P < 0.05). Post hoc tests showed that Dbh −/− mice. Instead, quinpirole tended to have an 2014 Society for the Study of Addiction Addiction Biology Cocaine response in DBH mice β-arrestin/actin ratio Dbh –/– βArr2 Dbh –/– βArr2 10 20 30 40 50 60 70 80 90 100 110 120
Figure 2 β-arrestin2 overexpression in the nucleus accumbens restores normal cocaine sensitivity to Dbh −/− mice. (a) Representative
picture of βArr2 overexpression in the NAc of a Dbh −/− mouse that received the βArr2 virus. (b) Western blot data (mean ± SEM above,
representative blot below) for βArr2 : actin ratio in the NAc of Dbh −/− mice that received GFP virus (n = 6) or βArr2 virus (n = 7).
***P < 0.001 compared with Dbh −/− with GFP virus. (c) Novelty-induced (drug-free state) and (d) cocaine-induced (15 mg/kg, i.p.,
administered after 30 minutes in chamber) locomotor activity in Dbh +/− mice (n = 8), Dbh −/− mice that received GFP virus (n = 10), and Dbh
−/− mice that received βArr2 virus (n = 11). *P < 0.05, **P < 0.01 compared with Dbh +/− controls. AC = anterior commissure; core = NAc
core; shell = NAc shell; arrow = needle track
excitatory effect in the knockout neurons, but it did not abolished the quinpirole-induced decrease in excitability quite reach significance [χ2 (d.f. = 1) = 3.5, P = 0.06] [χ2 (d.f. = 1) = 1.2, P = 0.27] and resulted in a significant (Fig. 4b & c). There was a highly significant difference difference in the spike frequency difference curve com- between the spike frequency difference curves between pared with the spike frequency difference curve for MSNs MSNs from controls compared with MSNs from Dbh −/− treated with quinpirole alone [χ2 (d.f. = 1) = 16.5; animals [χ2 (d.f. = 1) = 20.99, P < 0.0001] (Fig. 4c), P < 0.0001] (Fig. 5a & c). By contrast, the spike fre- indicating the excitability of MSNs is increased in Dbh −/− quency difference curve obtained from Dbh −/− MSNs animals compared with controls.
after application of NF023 was not significantly different Because D2-like receptor abundance is normal in Dbh from the spike frequency difference curve obtained −/− mice but the cellular response to quinpirole is altered, from Dbh −/− MSNs treated with quinpirole alone we suspected that D2 receptors might be aberrantly [χ2 (d.f. = 1) = 1.57, P = 0.21] (Fig. 5b & d).
coupled in NAc MSNs of Dbh −/− mice. To test this idea, Application of the Gαs inhibitor, NF449, did not we assessed the effects of quinpirole on MSN excitability occlude the inhibitory effects of D2 activation in MSNs in the presence of either a Gαi (NF023; 10 μM) or a from control mice; no changes in spike frequency Gαs (NF449; 1 μM) inhibitor (Freissmuth et al. 1996; were observed when compared with quinpirole alone Hohenegger et al. 1998). As expected for a Gαi-coupled [χ2 (d.f. = 1) = 0.14, P = 0.71] (Fig. 6a & c). However, receptor like D2, application of NF023 to control MSNs application of NF449 in Dbh −/− MSNs significantly 2014 Society for the Study of Addiction Addiction Biology Meriem Gaval-Cruz et al. Figure 4 Quinpirole inhibits evoked firing of NAc MSNs from
Figure 3 Activation of D2 receptors similarly inhibits evoked firing
control, but not Dbh −/− mice. Example traces of a MSN from a of NAc MSNs from Dbh +/− and C57BL/6J mice. Example traces of control (a) and Dbh −/− (b) animal at baseline, and following bath a MSN from a Dbh +/− (a) and a wild-type C57BL/6J (b) animal at application of the D2/3 agonist, quinpirole (5 μM), while action baseline and following bath application of the D2/3 agonist, potentials were evoked with a series of current steps. Dashed line quinpirole (5 μM), while action potentials were evoked with a series indicates −70 mV. (c) Population data (mean ± SEM) showing the of current steps. Dashed line indicates −70 mV. (c) Population data effects of D2-like receptor activation on evoked spike frequency (mean ± SEM) showing the effects of D2 receptor activation on spike differences (quinpirole − baseline) from cells recorded from control frequency differences (quinpirole − baseline) from cells recorded (n = 46) and Dbh −/− (n = 15) mice. *Indicates significant difference from Dbh +/− (n = 22) and C57BL/6J (n = 24) mice (P < 0.05) between control and Dbh −/− population data 2014 Society for the Study of Addiction Addiction Biology Cocaine response in DBH mice Figure 5 Gαi inhibition abolishes quinpirole-mediated inhibition of MSN spike excitability in control mice, but has no effect in Dbh −/− mice.
Example traces from MSNs recorded from a control (a) and a Dbh −/− (b) animal at baseline or following bath application of quinpirole (5 μM)
with the Gαi inhibitor NF023 (10 μM) applied internally, while action potentials were evoked with a series of current steps. Dashed line
indicates −70 mV. (c) Population data (mean ± SEM) showing the effects of D2-like receptor activation on evoked spike frequency differences
(quinpirole − baseline) in the presence of vehicle (n = 46) or NF023 (n = 10) from cells recorded from control mice. (d) Population data
(mean ± SEM) showing the effects of D2-like receptor activation on evoked spike frequency differences (quinpirole − baseline) in the presence
of vehicle (n = 15) or NF023 (n = 17) from cells recorded from Dbh −/− mice. *Indicates significant difference (P < 0.05) between vehicle and
NF023 population data
reduced spike frequency compared with quinpirole alone including psychostimulant drugs of abuse (Weinshenker [χ2 (d.f. = 1) = 14.99, P = 0.0001] (Fig. 6b & d). Com- et al. 2002, 2008; Haile et al. 2003; Schank et al. 2006; bined, these results suggest that D2 receptors on NAc Gaval-Cruz et al. 2012). We identified a decrease in MSNs primarily couple to Gαi and suppress firing rate in βArr2 and an increase in ΔFosB in the NAc control mice, but that D2-G-protein coupling is altered following chronic genetic or pharmacological DBH and quinpirole-induced inhibition is lost in Dbh −/− NAc inhibition, and overexpression of βArr2 normalized cocaine responses in Dbh −/− mice. Slice electro-physiology experiments revealed that MSNs from control mice are inhibited by quinpirole in a Gαi- dependent manner, while the effects of quinpirole are Pharmacological and genetic DBH inhibition leads altered and become sensitive to Gαs blockade in Dbh −/− to behavioral hypersensitivity to dopaminergic drugs, 2014 Society for the Study of Addiction Addiction Biology Meriem Gaval-Cruz et al. Figure 6 Gαs inhibition has no effect on quinpirole-mediated inhibition in control mice, but restores quinpirole-mediated inhibition of MSN
spike frequency in Dbh −/− mice. Example traces from MSNs recorded from a control (a) and a Dbh −/− (b) animal at baseline or following
bath application of quinpirole (5 μM) with the Gαs inhibitor, NF449 (10 μM; slices pre-incubated in the Gαs antagonist for 1 hour prior to
recording, and then continuously exposed to NF449 throughout the recording process), while action potentials were evoked with a series of
current steps. Dashed line indicates −70 mV. (c) Population data (mean ± SEM) showing the effects of D2-like receptor activation on evokedspike frequency differences (quinpirole − baseline) in the presence of vehicle (n = 46) or NF449 (n = 11) from cells recorded from controlmice. (d) Population data (mean ± SEM) showing the effects of D2-like receptor activation on evoked spike frequency differences (quinpirole− baseline) in the presence of vehicle (n = 15) or NF449 (n = 22) from cells recorded from Dbh −/− mice. *Indicates significant difference(P < 0.05) between vehicle and NF449 population data Chronic DBH inhibition alters the abundance of DA
Weinshenker & Schroeder 2007), producing a compen- receptor signaling proteins in the NAc
satory up-regulation of D2 signaling and hypersensitivity to psychostimulants and quinpirole (Weinshenker et al.
Because DBH catalyzes the conversion of NE to DA in 2002, 2008; Schank et al. 2006). Indeed, neurotoxic noradrenergic neurons, DBH inhibition decreases NE ablation of brain NE neurons, which reduces NE without production, with a concomitant increase in tissue DA an increase in tissue DA, confers a similar pattern of drug et al.
responses (Harro et al. 2000; Weinshenker et al. 2008; Bourdelat-Parks et al. 2005). However, because NE pro- Nowak et al. 2009).
vides direct and indirect excitatory drives onto midbrain We originally reported increased high-affinity state D2 DA neurons, basal and stimulant-evoked DA overflow is receptors in the striatum of Dbh −/− mice, which we actually reduced in Dbh −/− mice (Schank et al. 2006; speculated might underlie the behavioral hypersensitivity 2014 Society for the Study of Addiction Addiction Biology Cocaine response in DBH mice of the knockouts to psychostimulants (Schank et al.
frequency in MSNs. By contrast, Gαs-coupled receptors, 2006). However, subsequent in vitro radioligand compe- such as D1, have a facilitatory effect on MSN responses tition experiments failed to confirm these results (Hu & Wang 1988; West & Grace 2002; Surmeier et al.
(Skinbjerg et al. 2010) (our unpublished data). The dis- 2010). Our electrophysiological recordings from NAc crepancy between studies measuring D2 affinity states neurons confirmed that the D2/3 agonist, quinpirole, may be due to some differences in the radioligands suppressed evoked MSN firing in slices from control mice.
and approaches employed, but more concerning was By contrast, the inhibitory effects of quinpirole were abol- our failure to observe a genotype difference in the abun- ished in Dbh −/− MSNs, and in fact there was a trend for dance of high-affinity state D2 receptors in vivo using quinpirole to be excitatory.
positron emission tomography imaging (Skinbjerg et al.
There are several potential explanations for this change in cellular response to D2/3 activation. For Because of these issues and inconsistencies, we sus- example, the involvement of βArr2 in receptor desensiti- pected that changes in downstream signaling molecules, zation and endocytosis suggests a possible contribution of rather than D2 receptor affinity state, were responsible for altered D2 trafficking and localization. In addition, cocaine hypersensitivity following chronic NE deficiency.
because βArr2 recruits cAMP-degrading phospho- Both genetic and pharmacological DBH inhibition pro- diesterase to the membrane upon receptor binding (Perry duced a decrease of βArr2 and an increase of ΔFosB in the et al. 2002; Kendall & Luttrell 2009), reduction of βArr2 NAc. ΔFosB is a transcription factor that is induced by could dampen inhibition and promote excitation by chronic exposure to drugs or other environmental stimuli potentiating cAMP abundance. The reduction in βArr2 and enhances behavioral responses to cocaine (Kelz et al.
could also affect neuronal firing by altering G-protein- 1999). We chose to pursue the contribution of βArr2 independent GSK3β/Akt signaling, although we did not because it is upstream of ΔFosB in the DA receptor detect any differences in these proteins in the NAc of Dbh signaling pathway, and because it had not been implicated −/− mice. It is also possible that the decrease in βArr2 and in cocaine-induced behaviors; locomotor activity and the altered quinpirole response are unrelated. Future conditioned place preference following cocaine adminis- experiments to determine whether loss of βArr2 directly tration are unchanged in βArr2 knockout mice (Bohn causes aberrant D2/3 signaling will help identify the et al. 2003). We found that viral-mediated overexpression of βArr2 in the NAc suppressed the cocaine hypersensi- Given that NF023 abolished quinpirole-induced inhi- tivity in Dbh −/− mice. This effect was not due to a general bition in MSNs from control mice, but had no effect on motor activity because ambulatory quinpirole response in Dbh −/− MSNs, while NF449 had behavior in a novel environment was unaffected by no effect on quinpirole-induced inhibition in control βArr2 overexpression, and cocaine-induced locomotion MSNs, but suppressed firing in the presence of quinpirole was normalized to, but not below, control levels. Because in Dbh −/− mice, it is possible that at least some D2 recep- we examined and used a non-selective CMV promoter to tors are coupled to Gαs instead of Gαi in Dbh −/− MSNs. In drive overexpression of βArr2, we cannot attribute its vivo Gαi-to-Gαs switching has been reported for μ-opioid effects on cocaine-induced locomotion specifically to receptors and CB1 cannabinoid receptors following changes in D2 signaling. Future experiments using D1- chronic agonist exposure (Wang et al. 2005; Paquette and D2-specific promoters will be required to delineate the et al. 2007), and a reduction in βArr2 promotes Gαs-to- importance of βArr2 in direct versus indirect pathway Gαi switching of β-adrenergic receptors (Baillie et al.
MSNs. The discrepancies between the βArr2-mediated 2003). However, these data are associated with several phenotypes in Dbh −/− and βArr2 knockout mice may be limitations and must be interpreted with caution.
due to the complete lack of global βArr2 in the βArr2 Quinpirole is an agonist of both D2 and D3 DA receptors, knockout mice versus the partial reduction of βArr2 and both subtypes are present in the NAc. Thus, the Gαs- specifically in the NAc of Dbh −/− mice.
to-Gαi switch could be solely or preferentially affectingone of these two subtypes. D3 receptors and D2-D3 heterodimer receptors have been reported to couple to Aberrant cellular responses to quinpirole in NAc MSNs
Gαq, and thus promote neuronal excitation. However, of Dbh −/− mice
increased D3-Gαq or D2/D3-Gαq heterodimer signaling is Because normal D2 autoreceptor function is preserved in unlikely to underlie quinpirole-induced excitation in Dbh Dbh −/− mice (Paladini, Beckstead & Weinshenker 2007), −/− MSNs for two reasons. First, Dbh −/− mice are hyper- we focused our attention on potential changes in D2 sensitive to quinpirole but not the preferential D2/D3 receptor signaling in accumbal MSNs. Activation of D2 heterodimer agonist SKF83959 (our unpublished data).
and other Gαi/o-coupled receptors typically inhibits Second, the altered response of Dbh −/− MSNs to evoked action potentials, reducing firing and spike quinpirole is blocked by the Gαs inhibitor NF449, which 2014 Society for the Study of Addiction Addiction Biology Meriem Gaval-Cruz et al. does not interfere with Gαq signaling. NF023 and NF449 providing the nepicastat, and C. Strauss for helpful are not totally selective for Gαi and Gαs; e.g. both com- editing of the manuscript. This work was supported by pounds are also P2X and P2Y receptor antagonists the National Institute of Drug Abuse, National Institute (Lambrecht 1996; Braun et al. 2001). A contribution of of Mental Health, and National Institute of Neurological purine/pyrimidine receptors is nevertheless unlikely Disorders and Stroke (NIDA grants DA017963 and given our results because, as a ligand-gated ion channel, DA027535 to D.W., DA25040 and DA015040 to M.G.C., would have a direct effect on membrane potential and and DA030530 to C.A.P.; NIMH grant MH079276 to firing rate, which we did not observe. NF023 and NF449 C.A.P.; and NINDS grant NS060658 to C.A.P.).
also had different effects in our different groups of animals, their inhibition which is inconsistent with these Disclosure/Conflict of Interest
drugs acting as purinergic/pyrimidinergic antagonists.
DW is coinventor on a patent concerning the use of selec- Finally, our methods did not allow us to distinguish tive DBH inhibitors for the treatment of cocaine depend- between D1 and D2 MSNs, which precludes assigning ence (US-201-0274303-A1; ‘Methods and Compositions direct effects of quinpirole and could also account for the for Treatment of Drug Addiction'). MGC, RBG, DJP, DEB, high variability in some of our experiments, particularly RCM, RAH, DJ and CAP declare no conflict of interest.
the ones involving Dbh −/− recordings. In addition, itwill be important to test alterations in D2-G-protein associations using other techniques such as co- immunoprecipitation. Unfortunately, D2 antibodies of MGC, RBG, CAP and DW participated in the research sufficient quality and specificity for this approach are not design. MGC, DJP and RCM conducted the Western blot experiments. MGC and DJP conducted the behavioral experiments. RBG conducted the electrophysiology experiments. DK conducted the statistical analysis of the electrophysiology experiments. RAH and DEB contrib- The consequences of chronic reduction in DBH function uted analytic tools. MGC, RBG, CAP and DW wrote the may be relevant to drug addiction. Non-selective DBH inhibitors, like disulfiram, have shown promise in human laboratory studies and clinical trials for the treatment of stimulant dependence (Gaval-Cruz & Weinshenker 2009), and a large phase II trial of nepicastat for cocaine Baillie GS, Sood A, McPhee I, Gall I, Perry SJ, Lefkowitz RJ, Houslay MD (2003) Beta-arrestin-mediated PDE4 cAMP NCT01704196). Genetic or chronic pharmacological phosphodiesterase recruitment regulates beta-adrenoceptor reduction of DBH activity enhances some interoceptive switching from Gs to Gi. Proc Natl Acad Sci U S A 100:940–945.
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